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Title: Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

Abstract

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.

Authors:
;  [1];  [2];  [1];  [2];  [3];  [1];  [3];  [3];  [3]
  1. Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States)
  2. Department of Obstetrics and Gynecology and Reproductive Sciences, Yale School of Medicine, P.O. Box 208063, New Haven, CT 06520-8063 (United States)
  3. (United States)
Publication Date:
OSTI Identifier:
22149252
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 422; Journal Issue: 1; Other Information: Copyright (c) 2011 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CARCINOMAS; CATTLE; CELL PROLIFERATION; HELA CELLS; HUMAN POPULATIONS; ONCOGENES; RECEPTORS

Citation Formats

Magaldi, Thomas G., Almstead, Laura L., Bellone, Stefania, Prevatt, Edward G., Santin, Alessandro D., Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028, DiMaio, Daniel, E-mail: daniel.dimaio@yale.edu, Department of Therapeutic Radiology, Yale School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, P.O. Box 208024, and Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028. Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation. United States: N. p., 2012. Web. doi:10.1016/J.VIROL.2011.10.012.
Magaldi, Thomas G., Almstead, Laura L., Bellone, Stefania, Prevatt, Edward G., Santin, Alessandro D., Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028, DiMaio, Daniel, E-mail: daniel.dimaio@yale.edu, Department of Therapeutic Radiology, Yale School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, P.O. Box 208024, & Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028. Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation. United States. doi:10.1016/J.VIROL.2011.10.012.
Magaldi, Thomas G., Almstead, Laura L., Bellone, Stefania, Prevatt, Edward G., Santin, Alessandro D., Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028, DiMaio, Daniel, E-mail: daniel.dimaio@yale.edu, Department of Therapeutic Radiology, Yale School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, P.O. Box 208024, and Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028. Thu . "Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation". United States. doi:10.1016/J.VIROL.2011.10.012.
@article{osti_22149252,
title = {Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation},
author = {Magaldi, Thomas G. and Almstead, Laura L. and Bellone, Stefania and Prevatt, Edward G. and Santin, Alessandro D. and Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028 and DiMaio, Daniel, E-mail: daniel.dimaio@yale.edu and Department of Therapeutic Radiology, Yale School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040 and Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, P.O. Box 208024 and Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028},
abstractNote = {Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.},
doi = {10.1016/J.VIROL.2011.10.012},
journal = {Virology},
number = 1,
volume = 422,
place = {United States},
year = {Thu Jan 05 00:00:00 EST 2012},
month = {Thu Jan 05 00:00:00 EST 2012}
}
  • In most cervical carcinoma cells the E6 and E7 genes of specific human papillomaviruses are transcribed from viral sequences integrated into host cell chromosomes. Glucocorticoids activate the promoter elements of various human papillomaviruses in transient-expression assays. The authors have analyzed the effect of dexamethasone on the transcription rate of human papillomaviruses 18 E6 and E7 genes integrated at different chromosomal sites in four cervical cancer cell lines. Dexamethasone led to an increase in the transcription rate of the integrated E6-E7 sequences in C4-1 and C4-2 cells but led to a decrease in SW 756 cells and did not affect themore » transcription rate in HeLa cells. It thus appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to steroid hormones. The growth rate of all dexamethasone-treated cell lines correlated consistently with the expression of the papillomavirus E6 and E7 genes, supporting their role in the maintenance of the proliferative phenotype of cervical carcinoma cells. Since human papillomaviruses are integrated into the host cell genome at variable, presumably randomly selected chromosomal loci, regulatory mechanisms that influence viral gene expression, and hence cell growth, may differ among cancers of independent clonal origin.« less
  • Highlights: {yields} Integration of HPV into host genome critical for activation of E6 and E7 oncogenes. {yields} BAF53 is essential for higher-order chromatin structure. {yields} BAF53 knockdown suppresses E6 and E7 from HPV integrants, but not from episomal HPVs. {yields} BAF53 knockdown decreases H3K9Ac and H4K12Ac on P105 promoter of integrated HPV 18. {yields} BAF53 knockdown restores the p53-dependent signaling pathway in HeLa and SiHa cells. -- Abstract: Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways,more » respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.« less
  • Highlights: • As{sub 2}O{sub 3} inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As{sub 2}O{sub 3} is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As{sub 2}O{sub 3}) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearlymore » elucidated, particularly in solid cancers. Our previous data showed that As{sub 2}O{sub 3} induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As{sub 2}O{sub 3} on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As{sub 2}O{sub 3} than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As{sub 2}O{sub 3} than HPV 16-positive CaSki and SiHa cells. After As{sub 2}O{sub 3} treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As{sub 2}O{sub 3} is a potential anticancer drug for cervical cancer.« less
  • The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expressionmore » of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.« less
  • Felis catus papillomavirus type 2 (FcaPV2) DNA is found in feline cutaneous squamous cell carcinomas (SCCs); however, its biological properties are still uncharacterized. In this study, we successfully expressed FcaPV2 E6 and E7 putative oncogenes in feline epithelial cells and demonstrated that FcaPV2 E6 binds to p53, impairing its protein level. In addition, E6 and E7 inhibited ultraviolet B (UVB)-triggered accumulation of p53, p21 and pro-apoptotic markers such as Cleaved Caspase3, Bax and Bak, suggesting a synergistic action of the virus with UV exposure in tumour pathogenesis. Furthermore, FcaPV2 E7 bound to feline pRb and impaired pRb levels, resulting inmore » upregulation of the downstream pro-proliferative genes Cyclin A and Cdc2. Importantly, we demonstrated mRNA expression of FcaPV2 E2, E6 and E7 in feline SCC samples, strengthening the hypothesis of a causative role in the development of feline SCC. - Highlights: • FcaPV2 E6 binds to and deregulates feline p53 protein. • FcaPV2 E7 binds to and deregulates feline pRb protein. • FcaPV2 oncogenes inhibit UVB-induced apoptosis. • FcaPV2 E6E7 and E7 increase the lifespan of primary cells. • FcaPV2 E2, E6 and E7 are expressed at the mRNA level in feline SCC in vivo.« less